A fixed physiologically active substance obtained by fixing a physiologically active substance, such as an enzyme, a coenzyme, a hormone, a receptor or an inhibitor to an insoluble carrier is widely utilized as a material for a bioreactor, a biosensor or affinity chromatography for preparation of chemical agents, foods and medicines as well as for clinical diagnosis or therapy. In this case, the fixed physiologically active substance may be in the form of either granules or membranes, both of which are being studied by many researchers in various groups.
The form of a fixed physiologically active substance to a porous granular carrier has been studied to the greatest extent, but it has been pointed out that the efficacy of the fixed physiologically active substance is reduced because of the restricted diffusibility caused by the non-stirring layer in the inside and periphery of the grain and, as the case may be, only a small part of the latent activity of the fixed substance can often be utilized in the fixed system. (For instance, see JP-B-58-35679. The term "JP-B" as used herein means an "examined published Japanese patent publication".) In order to overcome this defect, a reaction method has been developed where an enzyme or the like is fixed to a fine porous membrane and the membrane is permeated with an aqueous substrate solution under pressure.
For the purpose of widely employing such a membrane-fixed physiologically active substance to a bioreactor, a biosensor, a material for affinity chromatography, clinical diagnosis and the like, it is important for the non-specific interaction between the membrane carrier and the protein to be as small as possible. Therefore, polysaccharides such as cellulose and the like, which have a high hydrophilicity but are not electrically charged, are preferred as the material for the membrane carrier.
Some methods were already known for fixing an enzyme or other physiologically active substance to a cellulosic membrane by chemical bonding. For instance, JP-A-56-97235 (the term "JP-A" as used herein means an "unexamined published Japanese patent application"), JP-B-58-44357 and JP-B-62-32919 disclose a method of activating a cellulosic membrane with an oxidizing agent and then fixing an enzyme to the thus-activated membrane. British Patent No. 1,183,260 discloses a method of activating cellulose or a like membrane with a triazine derivative and then fixing an enzyme to the thus activated membrane.
In these known methods, a physiologically active substance-fixed cellulosic membrane could be obtained. However, these known methods still have some problems and are not satisfactory for practical use. Specifically, in the activation method using an oxidizing agent, it is considered that the aldehyde group formed on the carrier reacts essentially with an amino group in the physiologically active substance to form a Schiff base whereby the physiologically active substance is fixed to the carrier. However, the Schiff base formed is not stable so that the fixed physiologically active substance is often released from the carrier. Where a reducing agent such as NaBH.sub.4 or NaBH.sub.3 CN is to be used for the purpose of preventing the release of the fixed physiologically active substance, the physiological activity of the substance is lowered by the treatment with such reducing agent. As a means of omitting the second stage reaction, such as a reduction, after the fixation of the physiologically active substance, a method of fixing a physiologically active substance with a triazine derivative has been developed. According to the method, however, the fixation reaction speed is slow and a relatively long period of time is required for the fixation. Therefore, when a physiologically active substance which is unstable is to be fixed using this method, deactivation of the substance during the course of the fixation is a serious problem.
British Patent No. 1,183,260 indicates activation of a membrane carrier (such as filter paper or cotton fabric) with a triazine derivative where the membrane is immersed in an alkali substance. However, where the method is applied to an asymmetric membrane made of fine fibers, for example, a cellulosic asymmetric membrane (such as a microfilter), the membrane is noticeably deformed probably because of hydrolysis of cellulose and the strength of the membrane is decreased. This is a serious difficulty.
As a result, development of a method of activating a cellulosic membrane without decreasing the properties of the membrane itself, as well as a method of fixing a physiologically active substance to the cellulosic membrane thus activated so that it has high reactivity with protein is desired.